The ability of double stranded RNA to effectively silence gene expression, a phenomenon now commonly known as RNA interference (RNAi), has been one of the biggest scientific findings of the past decade. Recently, scientists Andrew Fire and Craig Mello were awarded the 2006 Nobel Prize for Medicine for their pioneering work in the RNAi field. However, many challenges still remain to move RNAi from the laboratory to the clinic. The biggest challenge to RNAi-mediated inhibition of target gene expression in animals and particularly humans is the efficient delivery of the RNAi agent to a sufficient number of target cells. A variety of delivery mechanisms are currently being explored in the RNAi field.
For instance, a number of groups have demonstrated successful and efficient delivery of double stranded (ds) RNA to mouse liver by tail vein injection. McCaffrey et al. (Nature Biotechnol. (2003) 21(6): 639-44) report inhibiting production of hepatitis B virus replicative intermediates in mice following tail vein injection of plasmids expressing HBV specific short hairpin RNAs (shRNAs). Giladi et al. (Mol. Therapy (2003) 8(5): 769-76) also report inhibition of HBV replication in mice following tail vein injection of HBV specific short interfering RNA (siRNA), and Song et al. (Nature Med. (2003) 9(3): 347-51) report RNA interference of fulminant hepatitis in mice following tail vein injection of siRNA specific for the fas gene.
While tail vein injection is suitable for inhibiting gene expression in mice, it is not a clinically relevant technique that may be used for humans. However, several groups have also shown successful delivery of dsRNA therapeutics without tail vein injection by systemic delivery using synthetic dsRNAs with improved stability. See Soutschek et al. Nature (2004) 432(7014): 173-8; see also Morrissey et al. Hepatol. (2005) 41(6): 1349-56. Local administration to the liver has also been demonstrated by injecting double stranded RNA directly into the circulatory system surrounding the liver using renal vein catheterization. See Hamar et al. PNAS (2004) 101(41): 14883-8. Still others have reported successful delivery of dsRNA and particularly siRNA using cationic complexes or liposomal formulations. See, e.g., Landen et al. Cancer Biol. Ther. (2006) 5(12); see also Khoury et al. Arthritis Rheumatol. (2006) 54(6): 1867-77.
In addition to injection into the circulatory system and lipid-based means of delivering dsRNA, several groups have reported the use of retroviral and adenoviral vectors for introducing dsRNA into mammals. For instance, Van den Haute et al. (Human Gene Therapy (2003) 14: 1799-1807) report lentiviral vector delivery of short hairpin (shRNAs) against the reporter enhanced GFP (EGFP) that were shown to knock down gene expression of EGFP in mouse brain up to six months after transduction. McCaffrey et al. (Abstract No. 039, Keystone Symposia on siRNAs and miRNAs, Apr. 14-19, 2004) report intravenous infusion of recombinant adenoviruses expressing HBV-specific shRNAs in HBV infected mice as a possible treatment approach against hepatitis virus infection in animals.
Thus, although some success has been shown using localized delivery, or by using systemic delivery of stabilized or complexed dsRNA, there is still a great need for in vivo RNAi delivery mechanisms that do not require specialized formulations or invasive delivery procedures. Furthermore, the present inventors have shown that DNA-based endogenous delivery of dsRNA is especially advantageous in allowing one to avoid the interferon/PKR response while providing a prolonged supply of expressed dsRNA. See US 2004/0152117, which is herein incorporated by reference in its entirety. Accordingly, there is a particular need for targeted delivery mechanisms for DNA-based RNAi expression vectors that do not require the use of viruses.
RNA interference was first discovered in the nematode C. elegans by Nobel prize laureates Andrew Fire and Craig Mello and their colleagues. See U.S. Pat. No. 6,506,559, which is herein incorporated by reference. In US '559, Fire and Mello et al. report that dsRNA-mediated inhibition showed a surprising ability to cross cellular boundaries. This observation has since been described as a phenomenon that is particular to nematodes or invertebrates, and corresponding modes of such RNAi trafficking in vertebrate organisms have been generally dismissed.
The present inventors have surprisingly discovered, however, that intramuscular, intradermal and subcutaneous delivery of expression constructs encoding dsRNA results in targeted inhibition of gene expression in vivo in the liver and potentially other organs and tissues of mammalian organisms. Without wishing to be bound by any theory, the inventors hypothesize that delivery of dsRNA to the liver from the muscle or skin, for example, may be mediated by extracellular vesicles (exovesicles) containing expressed RNA molecules such as dsRNA, antisense, miRNA, or mRNA or injected/introduced RNA molecules such siRNA, shRNA, etc. that bud from the surface of transfected muscle cells. The extrusion of such exovesicles has been demonstrated for several cell types including muscle.
There is evidence in the art that certain lectins are exported from muscle cells and myoblasts through evaginations of the cell membrane which pinch off to form extracellular vesicles called exovesicles. Such lectins including beta galectins are known to be on the surface of the extruded exovesicles. See Cooper and Barondes, Evidence of export of a muscle lectin from cytosol to extracellular matrix and for a novel secretory mechanism. J. Cell Sci. (1990) 110: 1681-91; see also Harrison and Wilson, The 14 kDa beta-galactoside binding lectin in myoblast and myotube cultures: localization by confocal microscopy, J. Cell Sci. (1992) 101(Pt. 3): 635-46. Extracellular vesicles have also been observed at the periphery of fibroblasts, which are present in high quantity in the dermal layer of the skin. See Mehul and Hughes, 1997, Plasma membrane targeting, vesicular budding, and release of galectin 3 from the cytoplasm of mammalian cells during secretion, J. Cell Sci. 110: 1169-78. There is also evidence that lectins and certain glycoproteins may be cleared from the circulation by specific receptors on the surface of liver cells. See, e.g., Park et al., The asialoglycoprotein receptor clears glycoconjugates terminating with sialic acid alpha 2,6GalNAc. PNAS (2005) 102(47): 17125-9; see also Nagaoka et al., Galectin receptors are known to be expressed on the surface of hepatocytes. Furthermore, betagalectin receptors have been shown to be expressed in a polarized manner on the sinusoidal side of the hepatocytes, “A quantitative analysis of lectin binding to adult rat hepatocytes cell surfaces”, In Vitro Cellular and Developmental Biology (1988) 24: 401-412; “Participation of a galectin-dependent mechanism in the hepatic clearance of tissue-type plasminogen activator and plasma kallikrein.” Thromb Res (2003) 108: 257-262.
Thus, the present inventors propose that cytosolic content including RNAs, e.g., mRNA, expressed siRNA/shRNA/miRNA, as well as injected/introduced siRNA/shRNA/miRNA, or possibly even transfected DNA present in the cytosol can be packaged within these exovesicles and be transported to distal sites such as the liver. Other mechanisms of transfer have not been ruled out. Whatever the mechanism, to the present inventors' knowledge, no one has recognized or proposed that intramuscular, intradermal or subcutaneous administration of dsRNA and particularly expressed dsRNA may be used in vivo in mammalian organisms as a therapeutic nucleic acid delivery mechanism for liver diseases as well as diseases affecting other distal organs and tissues.